Our long term objective is to understand the roles of regulatory carboxypeptidases in physiological and pathological processes. Membrane-bound carboxypeptidase (CP) M is an important regulator of the kallikrein- kinin system via its ability to generate des-Arg-kinin agonists of the G-protein coupled kinin B1 receptor (B1R). Kinins are important regulators of renal and cardiovascular function and inflammatory processes. The major objective is to investigate a novel allosteric protein-protein interaction between CPM and B1R that enhances receptor signaling. Specific Aim 1: To explore the interaction of CPM and B1R on the membrane and determine the residues important for their interaction. Hypothesis: positively charged residues on CPM's exterior bind to membrane phospholipids to orient CPM on the membrane and basal interaction with the B1R is mediated by its C-terminal domain whereas allosteric modulation is mediated by a surface near the catalytic domain. We will: (i) determine whether CPM and B1R directly interact. (ii) investigate the importance of the exterior positive charges on CPM for proper membrane orientation and binding to the B1R. (ii) determine the site(s) on the B1R that mediates its binding to CPM. (iii) determine the site(s) on CPM that interacts with the B1R. (iv) generate peptides to interfere with CPM/B1R interactions. Specific Aim 2: To elucidate the characteristics of the interaction between CPM and the B1R that results in allosteric modulation of receptor activation. Hypothesis: CPM enhances B1R function by binding to an allosteric site and substrate binding by CPM results in B1R conformational change and enhanced receptor activation. We will determine: (i) the role of CPM specificity and peptide substrate affinity on allosteric modulation of B1R activation and (ii) the effect of CPM allosteric modulation on B1R conformation. Specific Aim 3: To determine the role of CPM-mediated allosteric modulation of B1R activation in G-protein coupling, -arrestin interaction and downstream signaling and to determine the importance of CPM/B1R interaction in regulating endothelial barrier function. Our hypothesis is that CPM interaction with the B1R enhances all B1R signaling via basal interaction but further enhances Gq-coupled responses by allosteric modulation. Furthermore, disruption of CPM/B1R interaction will attenuate B1R-mediated endothelial barrier damage in response to B2R agonist. We will determine: (i) G-protein coupling mediated by CPM interaction with the B1R vs. agonist binding. (ii) the involvement of -arrestin-2 recruitment in B1R agonist activation vs. CPM mediated allosteric enhancement . (iii) the downstream signaling output from the B1R via Gq and Gi mediated pathways in response to agonist or CPM interaction. (iv) the role of CPM in allosteric modulation of B1R signaling in regulating endothelial/vascular permeability. These studies will elucidate a novel mechanism for allosteric enhancement of B1R signaling by CPM which also generates its ligand. Understanding how this process can be regulated could lead to the development of novel drugs to treat renal and cardiovascular diseases. PUBLIC HEALTH RELEVANCE: We are investigating the interaction of two proteins, carboxypeptidase M and B1 receptor, on the cell surface that results in increased production of regulatory molecules that affect cell and organ function. The B1 receptor is known to regulate kidney and cardiovascular function, especially under conditions of infection or inflammation. Understanding how the interaction of these proteins can be enhanced or blocked could lead to the development of novel drugs to treat renal and cardiovascular diseases.